We have continued the investigation of the structure, activation, and regulation of the Mg(II)-ATP-dependent phosphoprotein phosphatase. New methods of enzyme purification have been developed utilizing high performance liquid chromatography (HPLC). The enzyme from rabbit skeletal muscle has been purified approximately 17,000 fold to near homogeneity. The enzyme consists of a 38 KDa catalytic subunit and a 31 KDa regulatory subunit, which has historically been referred to as modulator or inhibitor-2. A 62 KDa polypeptide, which previously co-purified with enzyme activity, was separated with the new purification procedure. Densitometric scans of SDS gels indicate a l:l stoichiometry of the catalytic and regulatory subunits. This, however, assumes identical staining properties of the two subunits. Measurements based on the activity of the catalytic and regulatory (as an inhibitor) subunits have suggested a higher regulatory to catalytic subunit stoichiometry, approximately 2:1. The enzyme specific activity has been found to exhibit a concentration dependence. This is seen with kinase Fa at maximally activating concentrations and suggests an association/dissociation which alters the specific activity of the enzyme. The time course of phosphatase activation by kinase Fa and Mg(II)-ATP and of phosphate incorporation into the modulator subunit was further investigated. Initially phosphate is rapidly incorporated into the modulator subunit. As phosphatase activation proceeds, there is a decrease in the amount of phosphate incorporated. As the phosphatase activity levels off at a steady-state level of enzyme activation, the amount of phosphate incorporated into the modulator subunit plateaus at a low stoichiometry of approximately 0.1 mole Pi per mole. After activating the phosphatase, Mg(II) chelation with EDTA was used to block the activation reaction and allow measurement of the time course of inactivation. A first-order inactivation was observed with a tl/2 of 13-15 minutes, reflecting an isomerization of active enzyme back to its inactive form. The data for phosphatase inhibition by the type II regulatory subunit of cAMP-dependent protein kinase (see last year's report) was subjected to a computer fitting based on a single binding site assumption. The good fit that was obtained demonstrated that binding of RII to a single site on the phosphatase can account for the results.